Alzheimer's disease (AD) currently afflicts more than five million people in the US. Approximately 360,000 new cases of AD are diagnosed annually in the US. Under the current therapeutic environment the number of new cases is projected to rise more than 3-fold to about 1 million new cases per year by 2050. Thus, development of new drugs that prevent or delay the progression of the disease are desperately needed. Currently four approved drugs are available for the treatment of the symptoms of late-onset or sporadic AD, but these treatments do not prevent or delay the progression of the disease. Many new approaches to disease modifying therapies are under development, but their clinical outcome remain uncertain. After disappointing results from many amyloid-based clinical candidates, it is likely that therapeutics targeting just A alone will not address all of the pathogenic events in te disease. Therefore development of new approaches based on other targets such as the D664 cleavage is urgently needed to develop an optimal treatment for AD. The goal of this proposal is to identify inhibitors for this new target: the intracytoplasmic C-terminal proteolysis of APP at Asp664 (D664) site. We have previously shown that this C-terminal cleavage of APP leads to the production of highly cytotoxic peptide APP-C31. Recently we have shown that the entire N-terminal APP fragment from this cleavage APPneo fragment can be detected in cell lysates after treatment with certain statins or after serum withdrawal, and production of this N-terminal fragment correlates with cell death and can be measured as a biomarker of this cleavage. Our studies show that the D664 cleavage event occurs in human patients, and is increased in early stages of the diseases in comparison to age-matched controls; mutation of this site prevents cytotoxicity of the -C-terminal fragment (C99) as well as by A itself. Importantly, using a transgenic mouse model, if we genetically prevented the C-terminal APP proteolysis through a point mutation (Asp664 -> Ala), we blocked the features of Alzheimer's disease: the memory loss, synapse loss, the brain atrophy (shrinkage), the electrophysiological abnormalities, and the brain scarring (gliosis). We also see an increase in the sAPP/A42 ratios in the transgenic mouse brain with the D664A mutation. Our data thus argue that the D664 cleavage does indeed represent a novel and potentially important new therapeutic target in AD. We have developed an assay to screen for inhibitors of this APP proteolysis, and this new assay is adaptable to a high-throughput screening (HTS) format. Screening of a small CNS-focused library has already led to the identification of a promising inhibitor. In this proposal we outline a stepwise plan to identify and develop potent inhibitors of this target. In collaboration with Dr. Cosford we plan to screen the diverse Sanford- Burnham small-molecule library. Based on our preliminary data, the chance of successfully attaining the key goals of this proposal is high. This project fulfills an important gap, and would lead to identification of inhibitors for this APP processing pathway, and possibly a new class of therapeutics for AD.